ITRM20090081A1 - USE OF 90K PROTEIN INHIBITORS FOR THE PREPARATION OF A MEDICATION FOR THE CURE OR PREVENTION OF TUMORS. - Google Patents

Description

Use of 90K protein inhibitors for the preparation of a medicament for the treatment or prevention of cancer

The present invention relates to the use of 90K protein inhibitors for the preparation of a medicament for the treatment or prevention of tumors.

More particularly, the invention relates to the use of inhibitors of the 90K protein for the preparation of a medicament for the treatment or prevention of tumors such as carcinoma of the breast, ovary, lung, gastrointestinal tract, melanoma, lymphomas or other tumors that overexpress 90K.

The term tumor will be used below to refer to malignancy, also known as cancer.

Cancer is a disease characterized by the uncontrolled proliferation of transformed cells that invade nearby tissues and spread to other regions of the body through a process known as metastasis. Metastatic cancers lead to death in patients in a period ranging from a few months to a few years. The drugs most used in the treatment of tumors are cytotoxic chemotherapy (called antiblastic or simply chemotherapy). These drugs work by damaging DNA or inhibiting cell duplication, and thus cause nonspecific death of both cancer cells and normal cells in the process of replication. The lack of specific action of chemotherapeutics is the basis of the considerable toxicity that follows their administration.

In the last decade, basic scientific research has significantly increased knowledge on the molecular mechanisms responsible for the transformation and proliferation of cancer cells. This has led to the development of "targeted" or "targeted therapy" drugs, ie drugs designed to act in a specific way on cancer cells that show a specific molecular and / or functional alteration. However, even the "targeted therapies" are burdened by side effects and often manage to counteract tumor growth only temporarily. Despite scientific advances and the introduction of new and "targeted" chemotherapy agents into the clinic, cancer remains a difficult disease to cure, responsible for about 13% of deaths worldwide.

In light of the foregoing, the need to have new, more effective and possibly less toxic anticancer therapies is evident. In this context, our attention has focused on treatments aimed at inhibiting the action of a protein associated with tumors called 90K.

90K, also known as Mac-2 BP or LGALS3BP, is a complex, oligomeric molecule, composed of subunits of about 90,000 daltons, rich in glucose chains and with different functional domains [1-4]. The protein is mainly secreted by epithelial cells and is found in the blood and other biological fluids of man at a concentration of mg / ml [3, 5]. 90K is also localized in the extracellular matrix (ECM) where it binds to collagen and fibronectin [6]. Although not a "transmembrane" protein, 90K is often found associated with the lining membrane of cells [7, 8], where it specifically binds galectin-3 [2], galectin-1 and -7 [9 ], beta-1 integrins [6], kitenine [10] and endosyalin [11].

Due to its "multi-ligand" functions, 90K is involved in the adhesive processes that regulate tissue homeostasis, such as homotypic adhesion, i.e. between cells of the same type (for example between epithelial cells), heterotypic adhesion, i.e. between different cells (for example between epithelial cells on the one hand and fibroblasts, endothelial cells or blood cells on the other), and the adhesion between cells and ECM. Experimental evidence indicates that 90K plays an important role in the processes of growth and metastatic spread of tumors thanks to its ability to mediate adhesion phenomena. For example, when tumor cells kept in single-cell suspension in in vitro culture flasks are exposed to purified recombinant 90K, there is an increase in cell-cell adhesion (homotypic adhesion) with the formation of multicellular aggregates (see example 1). This phenomenon, due to the ability of 90K to bind residues of galectin-3 and galectin-1 present on adjacent cells [12, 13], may have particular relevance in neoplastic progression as it promotes the survival of tumor cells that have entered the bloodstream during metastasis process.

90K is also able to make cancer cells resistant to the action of chemotherapy. In fact, it is known that cell adhesion to ECM proteins protects cells from apoptosis induced by antiblastic drugs [14, 15]. It has been shown that the adhesion of lymphoma cells to 90K, through beta-1 integrins, confers resistance to the cytotoxic action of some chemotherapeutics, reducing the percentage of cells in apoptosis [16]. This 90K effect may explain what has been observed in the clinic in patients with non-Hodgikin lymphomas, where high blood levels of protein are associated with poor response to chemotherapy and reduced survival [16-18].

Furthermore, the secreted 90K is able to increase the production of enzymes involved in the degradation of the extracellular matrix, such as ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) [19] and MMPs (metalloprotease) [20], an essential event in the invasion processes and tumor metastasis [21-24]. The 90K-induced MMP-7 production appears to be mediated by interleukin (IL) -6, since antibodies to IL-6 inhibit this effect [20]. IL-6 is a pro-inflammatory and proangiogenic cytokine that promotes tumor growth and metastasis [25, 26]. The ability of 90K to stimulate IL-6 production has been demonstrated in blood mononuclear cells [27, 28] and recently also in bone marrow mesenchymal stem cells [29]. The latter effect was related to the propensity of neuroblastoma to metastasize to the bone [29].

90K was originally identified in human breast cancer cell culture medium [1]. Thanks to the development of a specific monoclonal antibody, called SP2, it was possible to study the levels of 90K in the blood and other biological fluids, as well as to evaluate its expression in tumor tissues [1-4]. This protein was particularly elevated in the blood of patients with various forms of cancer, such as breast, ovarian, lung, gastrointestinal tract, melanoma and lymphomas [5, 30] and in non-cancer patients affected from viral infections such as HBV or HCV hepatitis, and HIV infection [31, 32]. In HCV hepatitis, blood levels of 90K are related to the severity of the disease and the duration of the infection. Furthermore, elevated 90K levels are an independent predictor of failure to respond to treatment with interferon [32] or to combined treatment with peginterferon and ribavirin [33]. In HIV-infected patients, the amount of protein in the circulation is directly related to the stage of the disease, with higher levels in the more advanced stages [34]. Furthermore, elevated 90K levels at diagnosis are an independent prognostic factor of overt AIDS progression [35].

As for cancer patients, recipients of this patent, the blood levels of 90K were associated with the prognosis. In fact, patients with breast [36] or ovarian [37] cancer with higher blood levels of the protein have a shorter survival than patients with normal levels. Even in patients with lymphoma (Hodgkin or non-Hodgkin), high blood levels of 90K are associated with reduced survival and reduced sensitivity to chemotherapy treatments [16-18].

The unfavorable prognostic significance of 90K in cancer patients was also observed when the expression of the protein was evaluated in tumor tissue. In patients with non-small cell lung cancer [38] or breast cancer [39], the high expression of the protein in tumor cells (evaluated in immunohistochemistry) was found to be a predictor of reduced survival. Furthermore, in these patients, high tumor levels of 90K are predictive of metastatic spread of the disease, similar to what has been observed for high blood levels [36]. Another tumor that frequently expresses high levels of 90K is melanoma. In this tumor, 90K is associated with malignant transformation and assumes diagnostic value since normal melanocytes do not express 90K [40].

Overall, the experimental and clinical data indicate that 90K has a non-secondary role in the development and progression of tumors and, therefore, the availability of agents capable of inhibiting the activity of this protein could be of help in combating tumor disease.

The author of the present invention has now found that 90K represents a determining factor in tumor progression and by using specific inhibitors of the protein it is possible to regress the tumor. In particular, the monoclonal antibody SP2 was used as a 90K inhibitor. This antibody was obtained using the hybridoma technique [41], by immunization of BALB / c mice with proteins secreted in the culture medium of human breast cancer cells [1]. It is an IgG1 isotype antibody that specifically recognizes 90K. The antibody was used for the development of a kit for the immuno-enzymatic assay (ELISA) of 90K in biological fluids [5]. The antibody has been patented as a reagent for the in vitro determination of the 90K concentration for diagnosis and prognosis in patients with HIV infection (US patent 5298391). The murine hybridoma cell line, from which the SP2 antibody is purified, was deposited, on February 5, 1993, in the name of Stefano Iacobelli, at the DSMZ (DEUTSCHE SAMMLUNG VON MIKROORGANISMEN UND ZELLKULTUREN GmbH), under the Treaty of Budapest , under the access number DSM ACC2116, Mascheroder Weg 1 B D-3300 Braunschweig, Germany, and at the C.N.C.M. (Collection Nationale de Cultures de Microorganismes) of the Pasteur Institute in Paris, France, under the access number I-1083.

Therefore, the specific object of the present invention is the use of inhibitors of the 90K protein for the preparation of a medicament for the prevention and / or treatment of tumors. In particular, the antibodies are selected from the group consisting of polyclonal, monoclonal antibodies, such as for example the monoclonal antibody is the SP2 antibody produced by the DSM ACC2116 hybridoma, bi-specific antibodies, single-chain or antibody fragments. The monoclonal antibody can be obtained by the hybridoma technique described by Koheler et al [41] or by recombinant DNA technique. Monoclonal antibodies can also be obtained from phage antibody libraries with the technique described by Clackson et al [42].

The tumors that can be treated are those that affect the human species such as breast, ovarian, lung, gastrointestinal tract carcinoma, melanomas, lymphomas, and other tumors that overexpress 90K. For these tumors, 90K has been shown to play a role in promoting tumor development and growth, and therefore inhibition of the protein could give certain benefits. However, it is not excluded that other tumors expressing high levels of 90K may benefit from the anti-90K treatment.

A further object of the present invention is the use of one or more inhibitors against the 90K protein, for example those defined above, for the diagnosis and characterization of tumors, both in vitro and in vivo. For this purpose, the antibody can be labeled with chromogens, fluorochromes, radioactive isotopes and used in immunohistochemical assays or for in vivo molecular imaging. The tumors of interest are, for example, but not exclusively, those selected from the group that invariably have high levels of expression of 90K such as carcinoma of the breast, ovary, lung, gastrointestinal tract, melanomas or lymphomas.

The present invention also relates to a composition comprising o consisting of the combination of one or more inhibitors of 90K, for example those defined above, and of one or more anticancer substances as active ingredients together with one or more pharmaceutically acceptable excipients and / or adjuvants. Among the anticancer substances we have experimental data on the association of the anti-90K SP2 antibody with docetaxel, but other drugs chosen from the group of chemotherapeutic agents can be used such as for example Paclitaxel, Anthracyclines, Fluoropyrimidines, vinca alkaloids, platinum derivatives or targeted therapy such as trastuzumab, bevacizumab, cetuximab, panitumumab, sunitinib, sorafenib, gefitinib, erlotinib.

The present invention will now be described by way of illustration, but not of limitation, according to its preferred embodiments, with particular reference to the figures of the attached drawings.

Figure 1 shows the homotypic aggregation of human melanoma cells following the addition of Figure 2 shows the formation of metastases following the inoculation of 90K in syngenic C57 / BL6 mice.

Figure 3 shows the inhibition by the SP2 antibody of the aggregation induced by 90K in human melanoma cells.

Figure 4 shows the changes in the size of tumors over time that grow in mice following the inoculation of melanoma cells, in the control group animals and in those treated with SP2 antibody.

Figure 5 shows the effect of SP2 treatment, alone or in combination with Docetaxel, on the growth of tumors derived from the human breast cancer cell line MDA-MD-231 in nude mice.

Example 1: Study of the role of 90K on homotypic adhesion of melanoma cells.

Materials and methods: A375 human melanoma cells are kept in single-cell suspension in PBS (0.5 ml) in prolipropylene tubes, at a concentration of 1x10 <6> cells / ml, and left to shake at 100 rpm, at 37 ° C , with or without purified recombinant 90K at a concentration of 10 µg / ml. After 1 hour, cell aggregation is stopped with the addition of 50 ml of 10% paraformaldehyde. The number of aggregates is calculated by difference by counting the remaining single cells under the microscope.

Results: In the presence of 90K there is a 60-70% increase in cell-cell adhesion (homotypic adhesion) with the formation of multicellular aggregates (Figure 1).

Example 2: In vivo study of the role of 90K in the formation of lung metastases from murine Lewis lung cancer cells.

Materials and methods: C57 / BL6 syngenic mice are inoculated intramuscularly, on the proximal part of the hind limb, with 50,000 Lewis murine lung cancer cells. One group remains as a control. On the other hand, one group is treated, after 3-4 days from inoculation, with 100 µg of 90K intravenously, for 5 consecutive days. After 20 days the mice are sacrificed and the number of metastases counted in the lungs fixed and stained with Bowen's solution (picric acid and formalin). Each group, control and treated, consists of 8 animals.

Results: Mice treated with 90K developed approximately twice as many metastases as controls, a statistically significant increase (Figure 2).

Example 3: Study of the role of the SP2 antibody on the homotypic adhesion induced by 90K of melanoma cells.

Materials and methods: A375 human melanoma cells are kept in single-cell suspension as in example 1, in the presence of purified recombinant 90K (10 µg / ml) and with the addition or not of SP2 at a concentration of 100 µg / ml. After 1 hour the cell aggregation is blocked and the aggregates counted as in example 2

Results: The addition of SP2 to A375 cells results in a significant reduction in cell aggregation (Figure 3).

Example 4: In vivo study of the role of the SP2 antibody on the tumor growth of melanoma cells.

Materials and methods: Nude mice are inoculated subcutaneously with 5x10 <6> human melanoma cells MEL 8863 (cells expressing 90K). One group remains as a control. On the other hand, one group is treated intraperitoneally with SP2 at a dose of 10 µg / kg twice a week. Tumor growth is monitored by measuring the diameters of the lesions up to 33 days after inoculation. Each group, control and treated, consists of 8 animals.

Results: The mice treated with SP2 developed tumors about 60% smaller than the control (0.4 cm <3> vs 1 cm <3>) (figure 4).

Example 5: In vivo study of the role of the SP2 antibody in association with docetaxel on the tumor growth of breast cancer cells.

Materials and Methods: Nude mice are inoculated subcutaneously with 5x10 <6> human breast cancer cells MDA-MD-231 (cells expressing 90K). One group remains as a control. Other groups are treated intraperitoneally with SP2 at a dose of 10 µg / kg twice a week or with control IgG at the same dose, or with Docetaxel at a dose of 7.5 mg / kg once a week or with the combination of SP2. and docetaxel. Tumor growth is monitored by measuring the diameters of the lesions up to 44 days after inoculation. Each group, control and treated, consists of 8 animals.

Results: Both the SP2 antibody and the docetaxel chemotherapy significantly reduce tumor growth compared to the control. This effect is greater when the two agents are used in combination (Figure 5).

BIBLIOGRAPHY

1. Iacobelli S, Arnò E, D'Orazio A, Coletti G. Detection of antigens recognized by a novel monoclonal antibody in tissue and serum from patients with breast cancer. Cancer Res. 1986; 46: 3005-10.

2. Koths K, Taylor E, Halenbeck R, Casipit C, Wang A, Cloning and characterization of a human Mac-2-binding protein, a newmember of the superfamily defined by the macrophage scavenger receptor cysteinerich domain, J Biol Chem 1993; 268: 14245-9.

3. Ullrich A, Sures I, D'Egidio M, Jallal B, Powell TJ, Herbst R, Dreps A, Azam M, Rubinstein M, Natoli C, et al. The secreted tumor-associated antigen 90K is a potent immune stimulator. J Biol Chem 1994; 269: 18401-7.

4. D'Ostilio N, Sabatino G, Natoli C, Ullrich A, Iacobelli S, 90K (Mac-2 BP) in human milk, Clin Exp Immunol 1996; 104: 543–6.

5. Iacobelli S, Arno E, Sismondi P, Natoli C, Gentiloni N, Scambia G, Giai M, Cortese P, Panici PB, Mancuso S. Measurement of a breast cancer associated antigen detected by monoclonal antibody SP-2 in sera of cancer patients. Breast Cancer Res Treat 1988; 11: 19-30.

6. Sasaki T, Brakebusch C, Engel J, Timpl R. Mac-2 binding protein is a cell-adhesive protein of the extracellular matrix which self-assembles into ringlike structures and binds b1 integrins, collagens and fibronectin. EMBO J 1998; 17: 1606 –1613.

7. Becker R, Lenter MC, Vollkommer T, Boos AM, Pfaff D, Augustin HG, Christian S. Tumor stroma marker endosialin (Tem1) is a binding partner of metastasisrelated protein Mac-2 BP / 90K. PHASEB J 2008; 22: 3059-67.

8. Wang Y, Ao X, Vuong H, Konanur M, Miller FR, Goodison S, Lubman DM. Membrane glycoproteins associated with breast tumor cell progression identified by a lectin affinity approach. J Proteome Res. 2008; 7: 4313-25.

9. Tinari N, Kuwabara I, Huflejt ME, Shen PF, Iacobelli S, Liu FT Glycoprotein 90K / MAC-2BP interacts with galectin-1 and mediates galectin-1-induced cell aggregation. Int J Cancer 2001; 91: 167-72.

10. Lee JH, Cho ES, Kim MY, Seo YW, Kho DH, Chung IJ, Kook H, Kim NS, Ahn KY, Kim KK Suppression of progression and metastasis of established colon tumors in mice by intravenous delivery of short interfering RNA targeting KITENIN, a metastasis-enhancing protein. Cancer Res 2005; 65: 8993–9003.

11. Becker R, Lenter MC, Vollkommer T, Boos AM, Pfaff D, Augustin HG, Christian S. Tumor stroma marker endosialin (Tem1) is a binding partner of metastasisrelated protein Mac-2 BP / 90K. PHASEB J 2008; 22: 3059-67.

12. Inohara H, Akahani S, Koths K, Raz A. Interactions between galectin-3 and Mac-2-binding protein mediate cell-cell adhesion. Cancer Res 1996; 56: 4530-4.

13. Grassadonia A, Tinari N, Iurisci I, Piccolo E, Cumashi A, Innominato P, D'Egidio M, Natoli C, Piantelli M, Iacobelli S. 90K (Mac-2 BP) and galectins in tumor progression and metastasis. Glycoconj J.

2004; 19: 551-6.

14. Damiano JS, Cress AE, Hazlehurst LA, Shtil AA, Dalton WS. Cell adhesion mediated drug resistance (CAM-DR): role of integrins and resistance to apoptosis in human myeloma cell lines. Blood 1999; 93: 1658.

15. Sethi T, Rintoul R, Moore S, et al. Extracellular matrix proteins protect small cell lung cancer cells against apoptosis: a mechanism for small cell lung cancer growth and drug resistance in vivo. Nat Med 1999; 5: 662.

16. Fornarini B, D’Ambrosio C, Natoli C, Tinari N, Silingardi V, Iacobelli S, Adhesion to 90K (Mac-2 BP) as a mechanism for lymphoma drug resistance in vivo, Blood 2000; 96: 3282–5.

17. Rea A, Calmieri G, Tinari N, Natoli C, Tagliaferro P, Morabito A, Grassadonia A, Bianco AR, Iacobelli S, 90K is a serum marker of poor prognosis in non-Hodgikin's lymphoma patients, Oncol Rep 1994; 723-5.

18. Zhang DS, Jiang WQ, Li S, Zhang XS, Mao H, Chen XQ, Li YH, Zhan J, Wang FH, Predictive significance of serum 90K / Mac-2BP on chemotherapy response in non-Hodgkin's lymphoma, Ai Zheng 22 , 870-3 (2003)

19. Canals F, Colomé N, Ferrer C, Plaza-Calonge Mdel C, Rodríguez-Manzaneque JC. Identification of substrates of the extracellular protease ADAMTS1 by DIGE proteomic analysis. Proteomics. 2006 Apr; 6 Suppl 1: S28-35.

20. Bair EL, Nagle RB, Ulmer TA, Laferté S, Bowden GT. 90K / Mac-2 binding protein is expressed in prostate cancer and induces promatrilysin expression. Prostate 2006; 66: 283-93.

21. Coussens LM, Werb Z. Matrix metalloproteinases and the development of cancer. Chem Biol. 1996 Nov; 3 (11): 895-904

22. Andreasen, P.A., Kjoller, L., Christensen, L., and Duffy, M.J. (1997). Int. J. Cancer 72, 1-22.

23. Fidler IJ. The pathogenesis of cancer metastasis: the 'seed and soil' hypothesis revisited. Nat Rev Cancer 2003; 3: 453-8.

24. Deryugina E, Quigley JP. Matrix metalloproteinases and tumor metastasis. Cancer Metast Rev 2006; 25: 9-34.

25. Huang SP, Wu MS, Shun CT, et al. Interleukin-6 increases vascular endothelial growth factor and angiogenesis in gastric cercinoma. J Biomed Sci. 2004; 11: 517-527.

26. Heikkilä K, Ebrahim S, Lawlor DA. Systematic review of the association between circulating interleukin-6 (IL-6) and cancer. Eur J Cancer. 2008 May; 44 (7): 937-45. Epub 2008 Apr 1

27. Powell TJ, Schreck R, McCall M, Hui T, Rice A, App H, Azam M, Ullrich A, Shawver LK.bA tumorderived protein which provides T-cell costimulation through accessory cell activation. J Immunother Emphasis Tumor Immunol. 1995 May; 17 (4): 209-21

28. Kalayci O, Birben E, Tinari N, Oguma T, Iacobelli S, Lilly CM. Role of 90K protein in asthma and TH2-type cytokine expression. Ann Allergy Asthma Immunol. 2004 Nov; 93 (5): 485-92

29. Fukaya Y, Shimada H, Wang LC, Zandi E, DeClerck YA. Identification of galectin-3-binding protein as a factor secreted by tumor cells that stimulates interleukin-6 expression in the bone marrow stroma. J Biol Chem. 2008 Jul 4; 283 (27): 18573-81. Epub 2008 May 1.

30. Natoli C, Ortona L, Tamburrini E, et al: Elevated serum levels of a 90,000 daltons tumorassociated antigen in cancer and in infection by human immunode®ciency virus (HIV). Anticancer Res 1994, 14: 1457-1460.

31. Natoli C, Iacobelli S, Ghinelli F. Unusually high level of a tumor-associated antigen in the serum of human immunodeficiency virus-seropositive individuals. J Infect Dis. 1991 Sep; 164 (3): 616-7

32. Artini M, Natoli C, Tinari N, Costanzo A, Marinelli R, Balsano C, Porcari P, Angelucci D, D'Egidio M, Levrero M, Iacobelli S. Elevated serum levels of 90K / MAC-2 BP predict unresponsiveness to alpha-interferon therapy in chronic HCV hepatitis patients. J Hepatol. 1996 Aug; 25 (2): 212-7

33. Iacovazzi PA, Cozzolongo R, Lanzillotta E, Frisullo S, Guerra V, Correale M. Serum 90K / Mac-2 binding protein (Mac-2BP) as a response predictor to peginterferon and ribavirin combined treatment in HCV chronic patients. Immunopharmacol Immunotoxicol.

2008; 30 (4): 687-700.

34. Natoli C, Dianzani F, Mazzotta F, Balocchini E, Pierotti P, Antonelli G, Iacobelli S. 90K protein: a new predictor marker of disease progression in human immunodeficiency virus infection. J Acquir Immune Defic Syndr. 1993 Apr; 6 (4): 370-5.

35. Briggs NC, Natoli C, Tinari N, D'Egidio M, Goedert JJ, Iacobelli S. A 90-kDa protein serum marker for the prediction of progression to AIDS in a cohort of HIV-1 + homosexual men. AIDS Res Hum Retroviruses.

1993 Sep; 9 (9): 811-6

36. Iacobelli S, Sismondi P, Giai M, D'Egidio M, Tinari N, Amatetti C, Di Stefano P, Natoli C, Prognostic value of a novel circulating serum 90K antigen in breast cancer, Br J Cancer 69, 172–6 (1994) 37. Zeimet AG, Natoli C, Herold M, Fuchs D, Windbichler G, Daxenbichler G, Iacobelli S, Dapunt O, Marth C, Circulating immunostimulatory protein 90K and soluble interleukin-2-receptor in human ovarian cancer, Int J Cancer 68, 34-8 (1996).

38. Marchetti A, Tinari N, Buttitta F, Chella A, Angeletti CA, Sacco R, Mucilli F, Ullrich A, Iacobelli S, Expression of 90K (Mac-2 BP) correlates with distant metastasis and predicts survival in stage I non- small cell lung cancer patients, Cancer Res 62, 2535–9 (2002) 39. Tinari N, Lattanzio R, Querzoli P, Natoli C, Grassadonia A, Alberti S, Hubalek M, Reimer D, Nenci I, Bruzzi P, Piantelli M , Iacobelli S; National Interuniversity Consortium for Bio-Oncology (CINBO). High expression of 90K (Mac-2 BP) is associated with poor survival in node-negative breast cancer patients not receiving adjuvant systemic therapies. Int J Cancer. 2009 Jan 15; 124 (2): 333-8.

40. Cesinaro AM, Natoli C, Grassadonia A, Tinari N, Iacobelli S, Trentini GP. Expression of the 90K tumor-associated protein in benign and malignant melanocytic lesions. J Invest Dermatol. 2002 Jul; 119 (1): 187-90.

41. Kohler, G., and Milstein, C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature (Lond.), 256: 495-497, 1975

42. Clackson T, Hoogenboom HR, Griffiths AD, Winter G. Making antibody fragments using phage display libraries. Nature. 1991 Aug 15; 352 (6336): 624-8.

Claims (13)

CLAIMS 1) Use of one or more inhibitors of the 90K protein for the preparation of a medicament for the prevention and / or treatment of tumors. 2) Use according to claim 1, wherein said one or more inhibitors are selected from the group consisting of anti-90K antibodies. Use according to claim 2, wherein the antibodies are selected from the group consisting of polyclonal, monoclonal, bi-specific, single chain antibodies or antibody fragments. 4) Use according to claim 3 in which the monoclonal antibody is the SP2 antibody produced by the DSM ACC2116 hybridoma. 5) Use according to each of the preceding claims in which the tumors are selected from the group consisting of carcinoma of the breast, ovary, lung, gastrointestinal tract, melanoma, lymphoma. 6) Use of one or more inhibitors of the 90K protein for the in vitro diagnosis of tumors. 7) Use according to claim 6 wherein the inhibitors are those defined in each of claims 2 to 4. 8) Use according to each of claims 6-7, in which the inhibitor is labeled with chromogens, fluorochromes, radioactive isotopes. 9) Use according to each of claims 6-8, in which the tumors are selected from the group consisting of carcinoma of the breast, ovary, lung, gastrointestinal tract, melanomas, lymphomas. 10) Composition comprising o consisting of the combination of one or more inhibitors of 90K and of one or more anticancer substances as active ingredients together with one or more pharmaceutically acceptable excipients and / or adjuvants. 11. Composition according to claim 10 wherein the inhibitors are those defined in each of claims 2 to 4. 12) Composition according to each of claims 10-11, in which the anticancer substances are selected from the group consisting of docetaxel, Paclitaxel, Anthracycline, Fluoropyrimidine, vinca alkaloids, platinum derivatives, trastuzumab, bevacizumab, cetuximab, panitumumab, sunitibinib, sorafen , gefitinib, erlotinib. 13) Use of the composition as defined in each of claims 10-12 for the preparation of a medicament for the treatment and / or prevention of tumors.